Not Applicable
This invention relates generally to materials and methods for isolating nucleic acids, such as plasmid DNA, chromosomal DNA, total RNA, mRNA, viral DNA, viral RNA, or RNA/DNA hybrids from contaminants, such as proteins, lipids, cellular debris, or other nucleic acids. This invention relates, particularly, to solid phases, including magnetic or non-magnetic matrices and chromatographic stationary phases, which bind to a target nucleic acid under one set of solution conditions and release the target nucleic acid under another set of solution conditions. More particularly, this invention relates to mixed-bed solid phases comprising at least two different solid phases, wherein each solid phase in the mixture binds to and releases a target nucleic acid under different conditions.
Many molecular biological techniques such as reverse transcription, cloning, restriction analysis, amplification and sequencing require that nucleic acids used in the techniques be substantially free of contaminants capable of interfering with such processing or analysis procedures. Such contaminants generally include substances that block or inhibit chemical reactions, (e.g. substances that hybridize to nucleic acids, or substances that block or inhibit enzymatically catalyzed reactions, and other types of reactions used in molecular biological techniques), substances that catalyze the degradation or depolymerization of a nucleic acid or other biological material of interest, or substances which block or mask detection of the nucleic acid of interest. Substances of this last type can block or mask by providing a xe2x80x9cbackgroundxe2x80x9d indicative of the presence in a sample of a quantity of a nucleic acid of interest, (also referred to herein as a xe2x80x9ctarget nucleic acidxe2x80x9d) when the nucleic acid of interest is not, in fact, present in the sample. Contaminants also include macromolecular substances from the in vivo or in vitro medium from which a target nucleic acid is isolated, macromolecular substances such as enzymes, other types of proteins, polysaccharides, or polynucleotides, as well as lower molecular weight substances, such as lipids, low molecular weight enzyme inhibitors, oligonucleotides, or non-target nucleic acids. Contaminants can also be introduced into a target biological material from chemicals or other materials used to isolate the material from other substances. Common contaminants of this last type include trace metals, dyes, and organic solvents.
Obtaining target nucleic acid, which is sufficiently free of contaminants for molecular biological applications, is complicated by the complex systems in which the target nucleic acid is typically found. Such systems (e.g., cells from tissues, cells from body fluids such as blood, lymph, milk, urine, feces, semen, or the like, cells in culture, agarose or polyacrylamide gels, or solutions in which target nucleic acid amplification has been carried out) typically include significant quantities of contaminants from which the target nucleic acid of interest must be isolated before being used in a molecular biological procedure.
Endotoxins are particularly problematic contaminants in preparations of nucleic acids isolated from gram-negative bacilli. Generally speaking, an endotoxin is a lipopolysaccharide material found in the cell wall of most such bacilli, including Escherichia Coli (xe2x80x9cE. colixe2x80x9d). During lysis of bacterial cells, such as is done to release plasmid DNA from E. coli transformants, endotoxins are released into the lysate produced thereby. Endotoxin contamination in a nucleic acid sample can adversely limit the utility of the sample, particularly in applications, which are sensitive to such contamination. For example, the transfection efficiency of several different cultured eukaryotic cell lines, including HeLa, Huh7, COS7, and LMH, have been shown to be sharply reduced in the presence of endotoxins. Weber, M. et al. 1995, BioTechniques 19(6):930-939. Endotoxins have also been found to be toxic to primary human cells, such as primary human skin fibroblasts and primary human melanoma cells, in the presence of entry-competent adenovirus particles. Cotten, M. et al. 1994, Gene Therapy 1:239-246. Endotoxins have also been shown to produce striking pathophysiological reactions when introduced into animals, including high fever, vasodilation, diarrhea and, in extreme cases, fatal shock. Morrison, David C. 1987, Ann. Rev. Med. 38:417-32.
Endotoxins are not readily separated from nucleic acids, particularly from plasmid DNA. Endotoxins tend to form micelles, which have a similar density, size, and charge distribution to plasmid DNA on the outer surface of the endotoxin micelles. As a result, endotoxins co-purify with nucleic acids, particularly with plasmid DNA, in most nucleic acid isolation procedures used today. For example, endotoxins appear in the same band as the DNA-ethidium bromide complex in the cesium chloride gradients used to separate plasmid DNA from other materials in a bacterial lysate. Endotoxins also co-migrate and co-elute with plasmid DNA from size exclusion and from anion exchange resins.
Conventional protocols for isolating DNA or RNA from various types of cells, including bacteria, begin with the cell disruption steps. See, e.g. Chapter 2 (DNA) and Chapter 4 (RNA) of F. Ausubel et al., eds., Current Protocols in Molecular Biology, Wiley-Interscience, New York (1993). Conventional DNA isolation protocols generally entail suspending the cells in a solution and using enzymes and/or chemicals, gently to lyse the cells, thereby releasing the DNA contained within the cells into the resulting lysate solution. For RNA isolation, conventional lysis and solubilization procedures include measures for inhibition of ribonucleases and contaminants, including DNA, to be separated from the RNA.
Many conventional procedures for isolating target nucleic acids from various mixtures of the target nucleic acids and contaminants, including mixtures produced from cells as described above, entail the use of hazardous chemicals such as phenol, chloroform, and ethidium bromide. For example, phenol or an organic solvent mixture containing phenol and chloroform are used in many such conventional procedures to extract contaminants from mixtures of target nucleic acids and various contaminants. Alternatively, cesium chloride-ethidium bromide gradients are used in place of or in addition to phenol or phenol-chloroform extraction. Closed circular DNA, such as plasmid DNA, intercalates with ethidium bromide and forms a band in a cesium chloride gradient formed after several hours of ultracentrifugation. The DNA/ethidium bromide band is extracted therefrom and the plasmid DNA isolated from the ethidium bromide using butanol or other conventional means. See, e.g., Molecular Cloning, ed. by Sambrook et al. (1989), pub. by Cold Spring Harbor Press, pp. 1.42-1.50. The phenol/chloroform extraction step, or cesium chloride banding and ethidium bromide extraction step is generally followed by precipitation of the nucleic acid material remaining in the extracted aqueous phase by adding ethanol to that aqueous phase. The precipitate is typically removed from the solution by centrifugation, and the resulting pellet of precipitate is allowed to dry before being resuspended in water or a buffer solution for further processing or analysis.
Such conventional nucleic acid isolation procedures have significant drawbacks. Among these drawbacks are the large amount of time required for multiple processing and extraction steps, and the dangers of using phenol and/or chloroform. Phenol causes severe burns on contact. Chloroform is highly volatile, toxic, and carcinogenic. Those characteristics require that phenol be handled and phenol/chloroform extractions be carried out in a fume hood. Another undesirable characteristic of phenol/chloroform extractions is that the oxidation products of phenol can damage nucleic acids. Only freshly redistilled phenol can be used effectively, and nucleic acids cannot be left in the presence of phenol. Generally also, multi-step procedures are required to isolate RNA after phenol/chloroform extraction. Ethanol (or isopropanol) precipitation must be employed to precipitate the DNA from a phenol/chloroform-extracted aqueous solution of DNA and remove residual phenol and chloroform from the DNA. Further, ethanol (or isopropanol) precipitation is required to remove some nucleoside triphosphate and short (i.e., less than about 30 bases or base pairs) single or double-stranded oligonucleotide contaminants from the DNA. Moreover, under the best circumstances such methods produce relatively low. yields of isolated nucleic acid material, and the isolated nucleic acid material is contaminated with impurities.
Cesium chloride gradients take time to form, requiring at least four hours of spin time in even the fastest, most modem centrifuges. Ethidium bromide, required for banding of plasmid or chromosomal DNA such gradients, is a mutagen. Also, when cesium chloride banding is used to isolate plasmid DNA from bacterial lysates without any preceding or succeeding protein extraction step, such as a phenol/chloroform extraction, the plasmid DNA isolated therewith has been found to be highly contaminated with endotoxins. See, e.g. Cotten, et al., Gene Therapy (1994) 1:239-146, at 240 [Table 1].
Several simpler, faster, and safer methods have been developed which utilize solid phases such as chromatographic resins or silica-based material to isolate nucleic acids from cell lysates or other mixtures of nucleic acids and contaminants. However, each such isolation system developed so far has its own unique drawbacks. Specifically, most such solid phase extraction systems either fail to eliminate undesirable contaminants, such as endotoxins, or they introduce undesirable contaminants not present in an initial nucleic acid mixture, such as proteases or corrosive salts. Each such contaminant must be removed using additional extraction steps before the nucleic acid isolated therewith can be used in applications sensitive to such contaminants.
One of the first solid phases developed for use in isolating nucleic acids was a specialized resin of porous silica gel particles designed for use in high performance liquid chromatography (HPLC). The surface of the porous silica gel particles was functionalized with anion-exchangers, which could exchange with plasmid DNA under certain salt and pH conditions. See, e.g. U.S. Pat. Nos. 4,699,717, and 5,057,426. Machrey-Nagel Co. (Dxc3xcren, Germany), one of the first companies to provide HPLC columns packed with such anion-exchange silica gel particles. Machrey-Nagel continues to sell such columns today. See, e.g. formation about NUCLEOGEN(copyright) 4000-7DEAE in product information downloaded from the Machrey-Nagel homepage on the Internet on Jun. 12, 1998, at http://www.machrey-nagel.com. Each such column was designed so that plasmid DNA bound thereto is eluted in an aqueous solution containing a high concentration of a highly corrosive salt (e.g. plasmid DNA is eluted from the NUCLEOGEN(copyright) 4000-7DEAE column in 6 M urea). Each such column had to be washed thoroughly between each isolation procedure to remove the corrosive salt and contaminants bound to the column with the DNA from the system. The nucleic acid solution eluted therefrom also had to be processed further to remove the corrosive salt therefrom before it could be used in standard molecular biology techniques, such as cloning, transformation, digestion with restrictive enzymes, or amplification.
Various silica-based solid phase separation systems have been developed since the early HPLC systems described above. Modem silica-based systems utilize controlled pore glass, filters embedded with silica particles, silica gel particles, resins comprising silica in the form of diatomaceous earth, glass fibers or mixtures of the above. Each modem silica-based solid phase separation system is configured to reversibly bind nucleic acid materials when placed in contact with a medium containing such materials in the presence of chaotropic agents. Such solid phases are designed to remain bound to the nucleic acid material while the solid phase is exposed to an external force such as centrifugation or vacuum filtration to separate the matrix and nucleic acid material bound thereto from the remaining media components. The nucleic acid material is then eluted from the solid phase by exposing the solid phase to an elution solution, such as water or an elution buffer. Numerous commercial sources offer silica-based resins designed for use in centrifugation and/or filtration isolation systems. See, e.g. Wizard(trademark) DNA purification systems products from Promega Corporation (Madison, Wis., U.S.A.); or the QiaPrep(trademark) DNA isolation systems from Qiagen Corp. (Santa Clarita, Calif., U.S.A.)
Magnetically responsive particles, formerly used to isolate and purify polypeptide molecules such as proteins or antibodies, have also been developed for use as solid phases in isolating nucleic acids. Several different types of magnetically responsive particles designed for isolation of such materials are described in the literature, and many of those types of particles are available from commercial sources. Such particles generally fall into either of two categories, those designed to reversibly bind nucleic acid materials directly, and those designed to reversibly bind nucleic acid materials through an intermediary. For an example of particles of the first type, see silica based porous particles designed to reversibly bind directly to DNA, such as MagneSil(trademark) particles to be made commercially available from Promega Corporation, or BioMag(copyright) magnetic particles available from PerSeptive Biosystems. For examples of particles and systems of the second type designed to reversibly bind one particular type of nucleic acid (mRNA), see the PolyATract(copyright) Series 9600(trademark) mRNA Isolation System from Promega Corporation; or the ProActive(trademark) line of streptavidin coated microsphere particles from Bangs Laboratories (Cannel, Ind., U.S.A.). Both of these latter two systems employ magnetically responsive particles with avidin subunits covalently attached thereto, and streptavidin with an oligo dT moiety covalently attached thereto. The streptavidin-oligo dT molecules act as intermediaries, hybridizing to the poly A tail of mRNA molecules when placed into contact therewith, then binding to the particles through a releasable streptavidin-avidin bond.
The indirect binding magnetic separation systems for nucleic acid isolation or separation all require at least three components, i.e. magnetic particles, an intermediary, and a medium containing the nucleic acid material of interest. The intermediary/nucleic acid hybridization reaction and intermediary/particle binding reaction often require different solution and/or temperature reaction conditions from one another. Each additional component or solution used in the nucleic acid isolation procedure adds to the risk of contamination of the isolated end product by nucleases, metals, and other deleterious substances.
Various types of magnetically responsive silica based particles have been developed for use as solid phases in direct or indirect nucleic acid binding isolation methods. One such particle type is a magnetically responsive glass bead, preferably of a controlled pore size. See, e.g. Magnetic Porous Glass (MPG) particles from CPG, Inc. (Lincoln Park, N.J., U.S.A.); or porous magnetic glass particles described in U.S. Pat. Nos. 4,395,271; 4,233,169; or 4,297,337. Nucleic acid material tendsto bind very tightly to glass, however, that it can be difficult to remove once bound thereto. Therefore, elution efficiencies from magnetic glass particles tend to be low compared to elution efficiencies from particles containing lower amounts of a nucleic acid binding material such as silica.
Another type of magnetically responsive particle designed for use as a solid phase in direct binding and isolation of nucleic acids, particularly DNA, is a particle comprised of agarose embedded with smaller ferromagnetic particles and coated with glass. See, e.g. U.S. Pat. No. 5,395,498. A third type of magnetically responsive particle designed for direct binding and isolation of nucleic acids is produced by incorporating magnetic materials into the matrix of polymeric silicon dioxide compounds. See, e.g. German Patent No. DE 43 07 262 A1. The latter two types of magnetic particles, the agarose particle and the polymeric silicon dioxide matrix, tend to leach iron into a medium under the conditions required to bind nucleic acid materials directly to each such magnetic particle. It is also difficult to produce such particles with a sufficiently uniform and concentrated magnetic capacity to ensure rapid and efficient isolation of nucleic acid materials bound thereto.
Silica-based solid phase nucleic acid isolation systems, whether magnetic or non-magnetic based or configured for direct or indirect binding, are quick and easy to use and do not require the use of corrosive or hazardous chemicals. However, such systems are ineffective at isolating nucleic acids from contaminants, such as endotoxins, which tend to bind to and elute from such solid supports under the same conditions as nucleic acids. See, e.g. Cotten, Matt et al. Gene Therapy (1994) 1:239-246.
Some nucleic acid isolation systems have been developed in which a nucleic acid solution containing proteins is pretreated with proteases to digest at least some of the proteins contained therein prior to isolation of the nucleic acid using a silica-based solid support of the type described above. See, e.g., QiaAmp(trademark) Blood Kit provided by QIAGEN Inc. (Santa Clarita, Calif.), which utilizes protease; and Wizard(copyright) Plus SV Minipreps DNA Purification System provided by Promega Corp. (Madison, Wis.), which utilizes an alkaline protease. However, such pre-treatment systems require the introduction of one contaminant into a mixture to digest another contaminant. Carry-over proteases can limit the utility of nucleic acids isolated using such modified silica-based systems at least as much as nucleic acid samples contaminated with the proteins the proteases are introduced to digest. Specifically, given the proper solution conditions, proteases in a nucleic acid solution will digest any proteins introduced into the solution, including enzymes introduced therein to modify, cut, or transcribe the nucleic acid contained therein for downstream processing or analysis. Protease addition, incubation and removal steps also drive up the cost of nucleic acid isolation, costing time and money compared to isolation systems with no such additional steps.
In all the solid phase systems described above, each solid phase used therein has a substantially uniform surface composition designed to bind to a nucleic acid of interest, in the form of a silica or silica gel surface, or in the form of a silica gel or polymer surface modified with chemical groups exhibiting anion exchanger activities. Bimodal and multimodal systems have also been developed, in which multiple columns each of which contains a solid phase modified with a different chemical group from the other columns in the system (e.g., Wheatley J. B., J. Chromatogr. (1992) 603: 273), or in which a single column is used with a single solid phase with at least two different chemical groups (e.g., patent ""680; Little, E. L. et al., Anal. Chem. (1991) 63: 33). Each of the chemical groups on the surface of the solid supports in the single column or multicolumn multimodal systems is configured to bind to different materials in whatever substrate is introduced into the system. Only a few such bimodal or multimodal column chromatography systems have been developed specifically for nucleic acid isolation (see, e.g. U.S. Pat. No. 5,316,680). Surface group combinations used in such solid phase systems include reverse phase, ion exchange, size exclusion, normal phase, hydrophobic interaction, hydrophilic interaction, and affinity chromatography. Such systems are designed such that only one of the surface groups binds a target species, such as a nucleic acid, while the other surface group(s) bind to and remove one or more non-target species in a mixture.
The bimodal and multimodal systems are far from simple, efficient alternatives to conventional organic or resin methods of nucleic acid isolation described above. Multi-column systems are inherently complex to run, as each column has requires a unique set of mobile phase conditions to bind and/or release the desired target or non-target species bound to the stationary solid phase of the system. Non-target species tend to block adjacent functional groups configured to bind to the target species, thus adversely affecting overall yield. Also, all the bimodal or multimodal systems are only designed to separate a target species from other species for which functional groups have affinity.
Materials and methods are needed which can quickly, safely, and efficiently isolate target nucleic acids which are sufficiently free of contaminants, particularly endotoxins, to be used in molecular biology procedures. The present invention addresses the need for materials and methods which provide a rapid and efficient means for isolating target nucleic acids from any mixture of target nucleic acids and contaminants, including lysates of gram-negative bacteria, thereby providing purified nucleic acids which can be used in a variety of biological applications, including transfection of cultured cells and in vivo administration of nucleic acids to organisms.
Briefly, in one aspect, the present invention is a mixed-bed solid phase designed for use in isolating a target nucleic acid from a mixture comprising the target nucleic acid and at least one contaminant. In a preferred practice, multiple contaminants are included in the mixture. The mixed-bed solid phase comprises at least two different solid phases, each of which has a capacity to bind and to release the target nucleic acid in the presence of different solution conditions.
The mixed-bed solid phase of the present invention preferably comprises a first solid phase and a second solid phase, wherein:
the first solid phase has a capacity to bind to the target nucleic acid when combined with the mixture in the presence of a first solution, and a capacity to release the target nucleic acid bound thereto in the presence of a second solution;
the second solid phase has a capacity to bind to the target nucleic acid when combined with the mixture in the presence of the second solution, and a capacity to release the target nucleic acid bound thereto in the presence of the first solution; and
the first solid phase and the second solid phase each have a capacity to release the target nucleic acid bound thereto in the presence of an elution buffer.
In another aspect, the present invention is a mixed-bed solid phase for isolating a target nucleic acid from a mixture comprising the target nucleic acid and at least one contaminant, the mixed-bed solid phase comprising first silica magnetic particles and second silica magnetic particles, wherein:
(a) the first silica magnetic particles have a capacity to bind to the target nucleic acid when combined with the mixture in the presence of a first solution, and a capacity to release the target nucleic acid bound thereto in the presence of a second solution;
(b) the second silica magnetic particles have a capacity to bind to the target nucleic acid when combined with the mixture in the presence of the second solution, and a capacity to release the target nucleic acid bound thereto in the presence of the first solution; and
(c) the first silica magnetic particles and the second silica magnetic particles all have the capacity to release the target nucleic acid bound thereto in the presence of an elution buffer.
In this aspect of the present invention, the first silica magnetic particles and second silica magnetic particles are either silica magnetic particles each comprising at least one ion exchange residue covalently attached thereto, or silica magnetic particles comprising a siliceous oxide coating and no anion exchange residue, respectively. The ion exchange residue is either an anion exchange residue, or an ion exchange residue which has a positive charge in the presence of the first or second solution, such that it can exchange with the target nucleic acid within that solution.
In another aspect, the present invention is silica magnetic particles, comprising: a plurality of first silica magnetic particles, each of which comprises a first silica magnetic particle covalently attached to a diethylamino (DEA) anion exchange residue. Such DEA ion exchange silica magnetic particles are particularly suitable for use as one of the solid phases in the mixed-bed solid phase of the present invention described above, and in the methods of isolating a target nucleic acid of the present invention described below.
The present invention is, also, a method of isolating a target nucleic acid from a mixture comprising the target nucleic acid and at least one contaminant, using a mixed-bed solid phase comprising a first solid phase and a second solid phase. This method comprises the steps of:
(a) providing the mixed-bed solid phase;
(b) combining the mixture with the mixed-bed solid phase in the presence of a first solution and permitting the target nucleic acid to bind to the first solid phase;
(c) separating the mixed-bed solid phase from the first solution;
(d) combining the mixed-bed solid phase with a second solution, and permitting the target nucleic acid to release from the first solid phase and bind to the second solid phase;
(e) separating the mixed-bed solid phase from the second solution; and
(f) combining the mixed-bed solid phase with the elution buffer, and permitting the target nucleic acid to release from the mixed-bed solid phase into the elution buffer.
This embodiment of the present invention preferably further comprises additional steps, wherein the mixed-bed solid phase is recombined and separated from the first solution and the second solution prior to being combined with the elution buffer in step (f).
The methods and materials of the present invention can be used to isolate target nucleic acids including, but not limited to plasmid DNA, total RNA, amplified nucleic acids, and genomic DNA from a variety of contaminants, including but not limited to agarose and components of a bacteria, animal tissue, and blood cells other than the target nucleic acid.
The mixed-bed solid phase and method of this invention enable one to isolate a variety of different types of target nucleic acids quickly and efficiently from a variety of difference substances, without the need to use hazardous chemicals (e.g. phenol, chloroform, ethidium bromide, or cesium chloride). Without intent to limit the scope of the present inventions, it is proposed herein that the first solid phase and second solid phase work more efficiently in the mixed-bed configuration of the present invention than either phase would if used to isolate a target nucleic acid alone, in tandem, or as separate functional groups of the same solid phase, for the following reasons.
A mixed-bed configuration enables one to transfer the target nucleic acid back and forth from the first solid phase to the second solid phase, thereby, further isolating the target nucleic acid from contaminants associated therewith, with each transfer step. When only one of the solid phases is used to isolate the target nucleic acid, considerably fewer of the contaminants are removed.
When used in tandem, the target nucleic acid is only bound to and released from each solid phase once, leaving at least one contaminants i.e. the target nucleic acid mixture. When two different functional groups having different affinities for the target nucleic acid are provided on the same solid phase, the target nucleic acid is not as efficiently bound and released therefrom as it is transferred from one solid phase to another using the mixed-bed solid phase in the method of the present invention. The mixed-bed solid phase produces surprising and unexpected results compared to known solid phase systems or other chromatographic separation techniques.
Target nucleic acids isolated using the mixed-bed solid phase or methods of the present invention are sufficiently free of contaminants to be suitable for use in additional processing, analysis, or even administration to mammals. Applications of the present mixed-bed solid phase to isolate nucleic acids from a variety of different media will become apparent from the detailed description of the invention below. Those skilled in the art of this invention will appreciate that the detailed description of the invention is meant to be exemplary only and should not be viewed as limiting the scope of the invention.